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Then cells were harvested by centrifugation (6,000 rpm, 15 min), resuspended in lysis buffer [Tris-HCl (100 mM, pH 8.0), NaCl (300 mM), imidazole (20 mM), TCEP (5 mM), and PMSF (1 mM)], and incubated on ice with 1 mg/mL lysozyme for 30 min